Multiple myeloma (MM) is incurable because the malignant stem cell escapes therapy. The objective of this proposal is to identify and characterize the myeloma stem cell. It incorporates the use of fresh patient tissue, novel hypotheses and sophisticated technology to define the stem cell(s) and the malignant clone in MM, with the ultimate aim of developing more effective therapy. The MM stem cell subset(s) will be identified as the set(s) of B lineage cells able to transfer the human MM clone to a mouse in vivo termed myelomagenic. The research plan will characterize the cell types that comprise the malignant B lineage hierarchy of MM, as defined by their expression of clonotypic IgH VDJ transcripts, in blood, bone marrow and in the G-CSF mobilized blood used for transplantation. For this aim the main methods will be RT-PCR, single cell RT-PCR and in situ RT-PCR together with immunofluorescence and cell sorting, to monitor the kinetics of each subset of the myeloma clone during the disease process. The analysis will include pre-B cells found in G-CSF mobilized blood, and pre- and post-switch B cells, and plasma cells in peripheral blood and in bone marrow at diagnosis, during treatment, in stable phase, in relapse, and after transplantation. G-CSF mobilized blood will be analyzed before and after cryopreservation to evaluate the extent to which clonotypic B cells are reinfused during autologous transplantation. The myelomagenic capacity of MM B lineage subsets in blood, bone marrow or mobilized blood will be determined using a pre-clinical model of MM, in which ex-vivo human myeloma cells engraft progressive myeloma to immunodeficient mice. The frequency and phenotype of MM stem cells in patients will be monitored at sequential time points throughout their disease, including in mobilized blood and after autologous transplantation. The time required for mice to progress to terminal disease will be evaluated for each B lineage subset found to have myelomagenic activity. The B subsets that mediate spread in this model will be identified. Those B lineage subsets with self renewal capability will be analyzed using a sequential cell transfer system. In the long term, this work will provide a well characterized pre-clinical model to evaluate novel therapies.